CN102872012B - Application of compound capable of inhibiting protein kinase - Google Patents

Application of compound capable of inhibiting protein kinase Download PDF

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CN102872012B
CN102872012B CN201210418212.2A CN201210418212A CN102872012B CN 102872012 B CN102872012 B CN 102872012B CN 201210418212 A CN201210418212 A CN 201210418212A CN 102872012 B CN102872012 B CN 102872012B
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alcohol
alkyl
compound
tetrahydrochysene
indazole
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CN102872012A (en
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刘阳
马正伟
邹强
阳泰
李丽梅
李敏惠
杨淑霞
王衍堂
李华
郑静
刘进
罗兴燕
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Chengdu Medical College
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Chengdu Medical College
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Abstract

The invention discloses a compound capable of inhibiting protein kinase and applications thereof. The compound disclosed by the invention can effectively inhibit the activity of protein kinase and inhibit multiplication, can inhibit multiplication of abnormally activated T cells, can be used for treating autoimmune diseases, malignant tumors and other diseases, and has notable therapeutic actions particularly on lymphomata, multiple myeloma, breast cancer, liver cancer, transplant rejection, irritability and rheumatoid arthritis, thus having favorable industrial application prospect.

Description

A kind of purposes of Profilin kinases compound
Technical field
The present invention relates to kinase whose compound of a kind of Profilin and uses thereof.
Background technology
Protein kinase (protein kinases) is also known as protein phosphorylation enzyme (protein phosphakinase).The enzyme of one class catalytic proteins phosphorylation reaction.On the amino acid residue that it can transfer to the γ-phosphoric acid on adenosine triphosphate (ATP) protein molecule some serine, threonine or tyrosine residue hydroxyl on, thus change protein, the conformation of enzyme and activity.The phosphorylation of protein is the important step of multi-signal pathway, and the important life of the interior major part of cell lives through journey and all be unable to do without protein phosphorylation.These enzymes are crucial factors regulating cell signal to comprise in cell proliferation and cell differentiation.
Protein kinase falls into 5 types: Protein Serine/threonine kinase, protein tyrosine kinase, protein groups histidine kinase, albumen tryptophan kinases and albumen aspartyl/glutamyl kinases.Protein kinase serves important function in the adjustment of cell processes and maintenance, all abnormal kinase is observed in numerous disease state, described morbid state comprises: malignant tumor, immune disease, cardiovascular disease, diabetes, infectious disease, arthritis and other immunologic derangement, nervous system, as senile dementia, A Mohaici disease AD etc., have now found that relevant to protein kinase more than 400 kinds of human diseasess.
Kinases inhibitor is the compound of a class Profilin kinase activity.According to the kind of protein kinase, can be divided into serine/threonine protein kitase inhibitor or tyrosine kinase inhibitor, the former again can according to site of action, be divided into three groups, one group acts on catalytic domain, and one group acts on regulatory region, and another group all has effect to regulatory region and catalytic domain.The correlational study of multiple protein inhibitors of kinases is had to report, as patent application: CN201110310167, CN200810028982 etc. at present.Numerous research report, kinases inhibitor may be used for treatment by the protein kinase-activated disease such as malignant tumor, arthritis caused, simultaneously, it can also stop the lymphocytic activation of T, therapeutical effect (Zhou Yiming is had to panimmunity disease, Deng, the progress of tyrosine protein kinase inhibitor, 6 volume 3 phases in 2000).Because the medical usage of kinases inhibitor is remarkable, therefore, the research of such medicine is seemed especially important.
Summary of the invention
The object of the present invention is to provide the kinase whose noval chemical compound of a class Profilin.Another object of the present invention is to the novelty teabag that this compounds is provided.
Particularly, the invention provides the compound shown in formula I and preparing the purposes in kinases inhibitor,
Formula I
R 1for-OH ,-H, alkyl ,=O, or-OR 7, wherein, R 7for the alkyl of C1-C5;
R 2for-H, alkyl ,-R 5-NH-R 6or-R 8oH, wherein, R 5for alkyl or alkenyl, R 6for alkyl, cycloalkyl, R 8for the alkyl of C1-C5;
R 3for the C6-10 aromatic radical of the aromatic radical of-H, alkyl, C6-10, the alkyl of replacement or replacement;
Or, R 1, R 2cycloalkyl, cycloalkanes ketone group or the cycloalkyl be substituted, cycloalkanes ketone group is formed together with the carbon atom that they connect;
Or, R 2, R 3cycloalkyl, cycloalkanes ketone group or the cycloalkyl be substituted, cycloalkanes ketone group is formed together with the carbon atom that they connect;
R 4for N, O or S;
R 9for-H or alkyl, R 10for the aromatic radical of C6-10;
Or, R 9, R 10the aromatic radical of the aromatic radical of C6-10 or the C6-10 of replacement is formed together with the carbon atom that they connect.
Further, described kinases inhibitor is serine/threonine protein kitase inhibitor or tyrosine kinase inhibitor.
Wherein, described kinases inhibitor is treatment malignant tumor, autoimmune disease, the heart
Blood vessel
The medicine of disease, diabetes, infectious disease, arthritis, immunologic derangement or senile dementia.
Further, described malignant tumor is multiple myeloma, lymphocytic chronic myeloid leukemia, granulocyte leukemia, lymphoma, hepatocarcinoma, pulmonary carcinoma, cancer of pancreas, colon cancer or breast carcinoma; Described autoimmune disease is rheumatoid type arthritis, myeloproliferative diseases, graft-rejection, asthma, lupus erythematosus, psoriasis, allergy or contact dermatitis.
Wherein, described compound is formula II compound, 2-(4-phenyl-2-thiazolyl)-4,5,6,7-tetrahydrochysene-2H-indazole-3-alcohol or 2-(6-methoxyl group-2-[4-morpholinodithio base)-4,5,6,7-tetrahydrochysene-2H-indazole-3-alcohol; Wherein, the structural formula of formula II compound is as follows
Formula II,
R 1for-OH ,-H, alkyl ,=O, or-OR 7, wherein, R 7for the alkyl of C1-C5;
R 2for-H, alkyl ,-R 5-NH-R 6or-R 8oH, wherein, R 5for alkyl or alkenyl, R 6for alkyl, cycloalkyl, R 8for the alkyl of C1-C5;
R 3for the C6-10 aromatic radical of the aromatic radical of-H, alkyl, C6-10, the alkyl of replacement or replacement;
Or, R 1, R 2cycloalkyl, cycloalkanes ketone group or the cycloalkyl be substituted, cycloalkanes ketone group is formed together with the carbon atom that they connect;
Or, R 2, R 3cycloalkyl, cycloalkanes ketone group or the cycloalkyl be substituted, cycloalkanes ketone group is formed together with the carbon atom that they connect;
R 4for N, O or S.
Further, R1 is-OH ,-H, alkyl ,=O;
R2 is-H, alkyl ,-R5-NH-R6, and wherein, R5 is alkyl or alkenyl; R6 is alkyl, cycloalkyl;
R3 is-H, alkyl;
Or, form cycloalkyl, cycloalkanes ketone group or the cycloalkyl be substituted, cycloalkanes ketone group together with the carbon atom that R1, R2 connect with them;
Or, form cycloalkyl, cycloalkanes ketone group or the cycloalkyl be substituted, cycloalkanes ketone group together with the carbon atom that R2, R3 connect with them;
R4 is N or S.
Further,
R 1for-OH ,-H, or-OR 7, described R 7for the alkyl of C1-C3;
R 2for alkyl or the-R of-H, C1-C5 8oH, described R 8for the alkyl of C1-C3;
R 3for the alkyl of the C1-C3 of the alkyl of-H, C1-C3, phenyl or halogen substiuted;
Or, R 2, R 3the cycloalkyl of C3-8 is formed together with the carbon atom that they connect;
R 4for N, O or S.
Further,
R 1for-OH, or-OR 7, described R7 is methyl;
R 2for-H or-R 8oH, described R 8for ethyl;
R 3for the methyl that methyl, phenyl or trifluoro replace;
Or, R 2, R 3the cycloalkyl of C5-C6 is formed together with the carbon atom that they connect;
R 4for N, O or S.
Further preferably, described compound is:
2-(1 hydrogen-benzothiazolyl-2 base)-4,5,6,7-tetrahydrochysene-2 hydrogen-indazole-3-alcohol,
2-(1 hydrogen-benzimidazolyl-2 base)-4,5,6,7-tetrahydrochysene-2 hydrogen-indazole-3-alcohol,
1-(1,3-benzothiazolyl-2 base)-1,5,6,7-tetrahydrochysene-4H-indazole-4-ketone,
1-(1,3-benzothiazolyl-2 base)-3,4-dimethyl-1 hydrogen-pyrazoles-5-alcohol,
2-(2-[4-morpholinodithio base)-2,4,5,6-tetrahydro cyclopentyl pyrazoles-3-alcohol,
1-(2-[4-morpholinodithio base)-3-(trifluoromethyl)-1H-pyrazoles-5-alcohol,
1-(2-[4-morpholinodithio base)-4-(2-ethoxy)-3-methyl isophthalic acid H-pyrazoles-5-alcohol,
2-(2-[4-morpholinodithio base)-4,5,6,7-tetrahydrochysene-2H-indazole-3-acetate,
2-(2-benzoxazolyl)-4,5,6,7-tetrahydrochysene-2H-indazole-3-alcohol or
1-(2-[4-morpholinodithio base)-3-phenyl-1H-pyrazoles-5-alcohol.
Present invention also offers the compound shown in formula I, its structural formula is as follows:
Formula I
R 1for-OH ,-H, alkyl ,=O, or-OR 7, wherein, R 7for the alkyl of C1-C5;
R 2for-H, alkyl ,-R 5-NH-R 6or-R 8oH, wherein, R 5for alkyl or alkenyl, R 6for alkyl, cycloalkyl, R 8for the alkyl of C1-C5;
R 3for the C6-10 aromatic radical of the aromatic radical of-H, alkyl, C6-10, the alkyl of replacement or replacement;
Or, R 1, R 2cycloalkyl, cycloalkanes ketone group or the cycloalkyl be substituted, cycloalkanes ketone group is formed together with the carbon atom that they connect;
Or, R 2, R 3cycloalkyl, cycloalkanes ketone group or the cycloalkyl be substituted, cycloalkanes ketone group is formed together with the carbon atom that they connect;
R 4for N, O or S;
R 9for-H or alkyl, R 10for the aromatic radical of C6-10;
Or, R 9, R 10the aromatic radical of the aromatic radical of C6-10 or the C6-10 of replacement is formed together with the carbon atom that they connect.
Further, described compound is formula II compound, 2-(4-phenyl-2-thiazolyl)-4,5,6,7-tetrahydrochysene-2H-indazole-3-alcohol or 2-(6-methoxyl group-2-[4-morpholinodithio base)-4,5,6,7-tetrahydrochysene-2H-indazole-3-alcohol; Wherein, the structural formula of formula II compound is as follows
Formula II,
R 1for-OH ,-H, alkyl ,=O, or-OR 7, wherein, R 7for the alkyl of C1-C5;
R 2for-H, alkyl ,-R 5-NH-R 6or-R 8oH, wherein, R 5for alkyl or alkenyl, R 6for alkyl, cycloalkyl, R 8for the alkyl of C1-C5;
R 3for the C6-10 aromatic radical of the aromatic radical of-H, alkyl, C6-10, the alkyl of replacement or replacement;
Or, R 1, R 2cycloalkyl, cycloalkanes ketone group or the cycloalkyl be substituted, cycloalkanes ketone group is formed together with the carbon atom that they connect;
Or, R 2, R 3cycloalkyl, cycloalkanes ketone group or the cycloalkyl be substituted, cycloalkanes ketone group is formed together with the carbon atom that they connect;
R 4for N, O or S;
Work as R 2, R 3cycloalkyl, the R of C6 is formed together with the carbon atom that they connect 1during for-OH, R 4be not S.
Further, R1 is-OH ,-H, alkyl ,=O;
R2 is-H, alkyl ,-R5-NH-R6, and wherein, R5 is alkyl or alkenyl; R6 is alkyl, cycloalkyl;
R3 is-H, alkyl;
Or, form cycloalkyl, cycloalkanes ketone group or the cycloalkyl be substituted, cycloalkanes ketone group together with the carbon atom that R1, R2 connect with them;
Or, form cycloalkyl, cycloalkanes ketone group or the cycloalkyl be substituted, cycloalkanes ketone group together with the carbon atom that R2, R3 connect with them;
R4 is N or S;
Work as R 2, R 3cycloalkyl, the R of C6 is formed together with the carbon atom that they connect 1during for-OH, R 4be not S.
Further preferably, described compound is:
2-(1 hydrogen-benzimidazolyl-2 base)-4,5,6,7-tetrahydrochysene-2 hydrogen-indazole-3-alcohol,
1-(1,3-benzothiazolyl-2 base)-1,5,6,7-tetrahydrochysene-4H-indazole-4-ketone,
1-(1,3-benzothiazolyl-2 base)-3,4-dimethyl-1 hydrogen-pyrazoles-5-alcohol,
2-(2-[4-morpholinodithio base)-2,4,5,6-tetrahydro cyclopentyl pyrazoles-3-alcohol,
1-(2-[4-morpholinodithio base)-3-(trifluoromethyl)-1H-pyrazoles-5-alcohol,
1-(2-[4-morpholinodithio base)-4-(2-ethoxy)-3-methyl isophthalic acid H-pyrazoles-5-alcohol,
2-(2-[4-morpholinodithio base)-4,5,6,7-tetrahydrochysene-2H-indazole-3-acetate,
2-(2-benzoxazolyl)-4,5,6,7-tetrahydrochysene-2H-indazole-3-alcohol or
1-(2-[4-morpholinodithio base)-3-phenyl-1H-pyrazoles-5-alcohol.
Experiment shows; the compounds of this invention can effectively Profilin kinase activity, suppress abnormal activation T cell propagation; can be used for disease such as treatment autoimmune disease, malignant tumor etc.; particularly lymphoma, multiple myeloma, breast carcinoma, hepatocarcinoma, graft-rejection, allergy and rheumatoid arthritis are had significant therapeutic effect, there is good prospects for commercial application.
Accompanying drawing explanation
Fig. 1 is on the impact of protein kinase activity
Fig. 2 is on the impact of graft-rejection
Fig. 3 is on the impact of Tardive allergy
Detailed description of the invention
The preparation of embodiment 12-(1 hydrogen-benzothiazolyl-2 base)-4,5,6,7-tetrahydrochysene-2 hydrogen-indazole-3-alcohol (being called for short compound 1)
Mixed material 2-Ketohexamethylene methyl formate (0.264mol), 2-hydrazinobenzothiazole (0.333mol), toluene 700mL, get acetic acid 1ml(acetic acid and raw material 2-Ketohexamethylene methyl formate mol ratio is about 0.07:1), in 1000mL reaction bulb, heat 120-125 DEG C of back flow reaction 5h(TLC and monitor reaction end).Reactant liquor evaporated under reduced pressure, residue adds 800mL ethyl alcohol recrystallization, filters, dry, obtains orange/yellow solid powder 36g, yield 50%, and measure through HPLC, compound purity reaches 98.22%, and the Structural Identification data of this compound are as follows:
ESI-MS:m/z 270(M-1)-
UV(MeOH)λmax=218nm;
1H NMR(DMSO-d6，600MHz)：δ8.01(d,1H,J=7.86Hz),7.78(d,1H,J=7.80Hz),,7.46(t,1H,J=7.6Hz)，7.33(t,1H,J=7.5Hz),2.50(m,2H),2.20(m,2H),1.73(m,2H)，1.67(m,2H)。
13C NMR(DMSO-d6,150MHz);δ162.3,154.6,153.3,148.9,132.2,126.9,124.3,122.6,121.0,102.5,22.3,22.2,21.7,18.7;ESI-MS:m/z 272[M+1]+;HRESIMS m/z calcd for C14H14N3OS[M+1]+272.0852,found 272.0849.
The preparation of embodiment 22-(2-[4-morpholinodithio base)-2,4,5,6-tetrahydro cyclopentyl pyrazoles-3-alcohol (being called for short compound 5)
Except the hexamethylene ketone ester replaced with raw material Ketocyclopentane ester in above-mentioned reaction, all the other are prepared with embodiment 1.The preparation of embodiment 31-(2-[4-morpholinodithio base)-3-(trifluoromethyl)-1H-pyrazoles-5-alcohol (being called for short compound 6)
By reaction equation mixed material ester (2g, 0.011mol), raw material hydrazine (2.2g, 0.013mol), toluene 70mL, acetic acid 0.1mL are in 150mL reaction bulb, and external heat 120-125 DEG C of back flow reaction 5h(TLC monitors reaction end).Reactant liquor evaporated under reduced pressure, residue adds 70mL ethyl alcohol recrystallization, filters, dry, obtains white solid powder 0.23g, yield 5%.The Structural Identification data of this compound are as follows:
1H NMR(DMSO-d6，600MHz)：δ8.08(d,1H,J=6.0Hz),7.94(d,1H,J=6.0Hz), 7.52(t,1H,J=6.0Hz)，7.43(t,1H,J=6.0Hz),5.95(s,1H),3.43(s,1H)。ESI-MS:m/z 284(M-1)-
The preparation of embodiment 41-(2-[4-morpholinodithio base)-4-(2-ethoxy)-3-methyl isophthalic acid H-pyrazoles-5-alcohol (being called for short compound 7)
By the preparation method of embodiment 3, after replacing reaction raw materials, be prepared into white solid powder 0.89g, yield 8%.The Structural Identification data of this compound are as follows:
1H NMR(DMSO-d6，600MHz)：δ8.02(d,1H,J=12.0Hz),7.80(d,1H,J=6.0Hz),7.47(t,1H,J=12.0Hz)，7.33(t,1H,J=6.0Hz),3.47(t,2H,J=6.0Hz),2.37(t,2H,J=6.0Hz),2.20(s,3H)。
ESI-MS:m/z 274(M-1)-
The preparation of embodiment 52-(6-methoxyl group-2-[4-morpholinodithio base)-4,5,6,7-tetrahydrochysene-2H-indazole-3-alcohol (being called for short compound 8)
According to the preparation method of embodiment 3, after replacing reaction raw materials, obtain blue powder 0.7g, yield 7.0%.
The Structural Identification data of this compound are as follows:
1H NMR(DMSO-d6，600MHz)：δ7.67(d,1H,J=6.0Hz),7.62(s,1H),7.05(d,1H,J=6.0Hz)，3.80(s,3H),2.19(s,2H),1.72(d,1H,J=6.0Hz),1.66(d,3H,J=6.0Hz)。
ESI-MS:m/z 300(M-1)-
The preparation of embodiment 62-(2-benzoxazolyl)-4,5,6,7-tetrahydrochysene-2H-indazole-3-alcohol (being called for short compound 10)
According to the preparation method of embodiment 3, after replacing reaction raw materials, obtain pale powder 0.1g, yield 2.5%.The Structural Identification data of this compound are as follows:
1H NMR(DMSO-d6，600MHz)：δ7.68(m,2H),7.33(m,2H),2.17(s,2H),1.72(m,4H)。
ESI-MS:m/z 256(M+1)+
The preparation of embodiment 72-(4-phenyl-2-thiazolyl)-4,5,6,7-tetrahydrochysene-2H-indazole-3-alcohol (being called for short compound 11)
According to the preparation method of embodiment 3, after replacing reaction raw materials, obtain pale powder 0.9g, yield 9.4%.The Structural Identification data of this compound are as follows:
1H NMR(DMSO-d6，600MHz)：δ11.69(s,1H),7.97(d,2H,J=6.0Hz),7.71(s,1H),7.43(t,2H,J=6.0Hz),7.33(t,1H,J=6.0Hz),2.53(t,2H,J=6.0Hz),2.19(s,2H),1.73(d,2H,J=6.0Hz),1.67(d,2H,J=6.0Hz)。
ESI-MS:m/z 298(M+1)+
The preparation of embodiment 81-(2-[4-morpholinodithio base)-3-phenyl-1H-pyrazoles-5-alcohol (being called for short compound 12)
According to the preparation method of embodiment 3, after replacing reaction raw materials, obtain white solid powder 0.81g, yield 8.2%.The Structural Identification data of this compound are as follows:
1H NMR(DMSO-d6，600MHz)：δ8.05(d,1H,J=6.0Hz),7.89(m,3H),7.50(m,4H)，7.38(t,1H,J=6.0Hz),6.10(s,1H)。
ESI-MS:m/z 292(M-1)-
The preparation of embodiment 92-(2-[4-morpholinodithio base)-4,5,6,7-tetrahydrochysene-2H-indazole-3-acetate (being called for short compound 9)
2-(1 hydrogen-benzothiazolyl-2 base)-4,5,6,7-tetrahydrochysene-2 hydrogen-indazole-3-alcohol (1.0g, 0.0037mol), Ac2O 20mL prepared by Example 1 is in 100mL reaction bulb, and back flow reaction 1h(TLC monitors reaction end).Reactant liquor evaporated under reduced pressure, residue adds 20mL ethyl alcohol recrystallization, filters, dry, obtains faint yellow solid powder 0.91g, yield 90%.The Structural Identification data of this compound are as follows:
1H NMR(DMSO-d6，600MHz)：δ8.02(d,1H,J=6.0Hz),7.85(d,1H,J=6.0Hz),,7.48(t,1H,J=6.0Hz)，7.38(t,1H,J=6.0Hz),2.64(t,2H,J=6.0Hz),2.47(s,3H),2.41(t,2H,J=6.0Hz),1.76(d,2H,J=6.0Hz)，1.68(d,2H,J=6.0Hz)。ESI-MS:m/z 314(M+1)+
Beneficial effect of the present invention is illustrated below by way of test example.
Test example 1 the compounds of this invention is on the impact of protein kinase activity
CTLL-2 cell is according to 1 × 10 7individual cells/well paves the end 6 orifice plate, and adds the compounds of this invention 1.IL-2 hunger was cultivated after 6 hours, and add IL-2, final concentration is 100U/ml, hatches 10min.Collecting cell, 600G, 5min are centrifugal removes supernatant, and PBS washing once, is got cell precipitation, added cell pyrolysis liquid cell lysis.Collect albumen to detect for Western-blot, kinases to be measured is phosphorylation Akt, phosphorylation JAK3 and phosphorylation STAT5.
Experimental result: result is see Fig. 1.
Result shows, prepared by the compounds of this invention 1(embodiment 1) can Profilin matter kinases Akt(serine/threonine protein kitase), JAK3(tyrosine kinase) and STAT5(intracellular signaling and activating transcription factor) phosphorylation, show that the compounds of this invention can effective Profilin kinase activity.
Test example 2 the compounds of this invention is on the impact of T cell
1, to Resting T cells toxicity
Method:
1. human peripheral blood single nucleus cell (peripheral blood mononuclear cell, PBMC) prepare: healthy personnel donate blood, utilize PBS equal proportion dilute blood, add 3mlPBMC separating medium in 10ml point end glass centrifuge tube after, tilt 45 DEG C slowly to add 6ml blood-PBS mixed liquor, the centrifugal 20min of 600g, draw and be rich in PBMC intermediate layer, PBS uses cell culture medium resuspended after washing 2 times.
2. purified T cell from PBMC: utilize MiltenylT cell magnetic bead purification kit to be separated from PBMC and obtain T cell, step is as follows: the PBMC cell suspension of above-mentioned preparation, and centrifugal 5 minutes of 500G, removes supernatant; PBS buffer 1(pH=7.2, containing 0.5%BSA, 2mM EDTA) add biotin labeling anti-CD14, CD16, CD19, CD36, CD56, CD123, and GlycophorinA antibody 30 μ l after 45 μ l are resuspended, mix latter 4 DEG C and hatch 20 minutes.Add above-mentioned buffer 1ml 500G, 5 minutes washed cells, remove supernatant.Re-suspended cell, installs adsorption column, allows cell suspension by adsorption column, and absorption non-T cell, obtains purified T cell.
3.T born of the same parents are according to 2 × 10 5individual cells/well, spreads 96 orifice plates, and volume is 200 μ l.Compound maximum concentration is 50 μMs, according to 5 times of gradient dilutions, cultivates after 72 hours, uses CCK-8 staining detection of drugs to T cell toxicity.Cell survival rate=medicine group cell OD/ does not add medicine group cell quantity × OD.Survival rate is 95% be considered as not having toxicity to T cell with drug level.IC50 is the concentration of the active half of Drug inhibition T cell.Result is see table 1.
2, the two counteirritantia human T-cell's propagation of CD3, CD28 is suppressed
Method:
1. preparation is containing the dual anti-culture medium of anti-human CD3, CD28: wrap by after 12 hours containing anti-CD3antibody 2 μ g/ml PBS, after PBS washs 2 times, add the culture medium 100 μ l containing the anti-human CD28 antibody of 1 μ g/ml.
2. Carboxyfluorescein diacetate succinimidyl ester (CFSE): add in the middle of CFSE to the good T cell of purification, wherein CFSE final concentration is 2.5 μMs, and T cell concentration is 1 × 10 6individual/ml.37 ° of C hatch 10 minutes, and centrifugal 5 minutes of 300G, abandons supernatant, add after culture medium washs 2 times, with resuspended rear paving 96 orifice plate of the culture medium of 1 μ g/ml anti-human CD28 antibody.Compound adds according to after gradient dilution, cultivates 72 hours flow cytometer and observes, and calculates the IC50 of suppressor T cell propagation.Experimental result: result is see table 1.
Table 1
Note: " NA " represents that unrestraint is active or activity is extremely low.
The abnormality proliferation of T cell, has material impact to the generation of autoimmune disease and malignant tumor.Experimental result shows, the compounds of this invention significantly can suppress human activated t-cells, but does not have toxicity to Resting T cells.Illustrate that the autoimmune disease that the abnormality proliferation of the compounds of this invention to T cell causes and malignant tumor have certain prevention or therapeutical effect.
Test example 3 the compounds of this invention suppresses human lymphocyte tumor cell proliferation
Method: Jurkat cell paves the end 96 orifice plate according to 1500 cells/well.In cultivating system, compound maximum concentration is 50 μMs, does drug level dilution according to 5 times of gradients.Compound effects adds 10 μ l CCk-8 after 48 hours, after hatching 6h, utilize microplate reader to measure 450nM wavelength absorption value.
Compound on tumor inhibitory rate of cell growth computational methods are according to National Cancer Institute (National Cancer Institute, NCI) standard method is carried out: when Ti(medicine group cultivates 48h, CCK-8 colour developing absorbs OD value) >=Tz(CCK-8 colour developing absorption OD value when drug containing group is not cultivated initial), tumor cell survival=[(Ti-Tz)/(C-Tz)] × 100, wherein C be not drug containing group after 48 hours CCK-8 colour developing absorb OD value; As Ti<Tz, tumor cell survival=[(Ti-Tz)/Tz] × 100.
Experimental result: result is see table 2.
Table 2
Note: " NA " represents that unrestraint is active or activity is extremely low.Compound number is see test example 2.
Experimental result shows, the compounds of this invention is obvious to lymphocytoma cyto-inhibition, and wherein, the inhibitory action of compound 1,2,3 is more excellent.
Test example 4 the compounds of this invention suppresses people's multiple myeloma cells propagation
Method: multiple myeloma cells RPMI8226 cell paves the end 96 orifice plate according to 5000 cells/well.In cultivating system, compound maximum concentration is 50 μMs, does drug level dilution according to 5 times of gradients.Compound effects adds 10 μ l CCk-8 after 48 hours, after hatching 6h, utilize microplate reader to measure 450nM wavelength absorption value.Drug on tumor inhibitory rate of cell growth computational methods are according to National Cancer Institute (National Cancer Institute, NCI) standard method is carried out: when Ti(medicine group cultivates 48h, CCK-8 colour developing absorbs OD value) >=Tz(CCK-8 colour developing absorption OD value when drug containing group is not cultivated initial), tumor cell survival=[(Ti-Tz)/(C-Tz)] × 100, wherein C be not drug containing group after 48 hours CCK-8 colour developing absorb OD value; As Ti<Tz, tumor cell survival=[(Ti-Tz)/Tz] × 100.
Experimental result: result is see table 3.
Table 3
Compound number is see test example 2.
Experimental result shows, the compounds of this invention is obvious to multiple myeloma cells inhibitory action.Test example 5 the compounds of this invention suppresses human breast carcinoma tumor cell proliferation
Method: human breast carcinoma tumor cell MCF-7 cell paves the end 96 orifice plate according to 3000 cells/well.In cultivating system, maximum drug concentration is 50 μMs, does drug level dilution according to 5 times of gradients.Drug effect adds 10 μ l CCk-8 after 48 hours, after hatching 6h, utilize microplate reader to measure 450nM wavelength absorption value.Drug on tumor inhibitory rate of cell growth computational methods are according to National Cancer Institute (National Cancer Institute, NCI) standard method is carried out: when Ti(medicine group cultivates 48h, CCK-8 colour developing absorbs OD value) >=Tz(CCK-8 colour developing absorption OD value when drug containing group is not cultivated initial), tumor cell survival=[(Ti-Tz)/(C-Tz)] × 100, wherein C be not drug containing group after 48 hours CCK-8 colour developing absorb OD value; As Ti<Tz, tumor cell survival=[(Ti-Tz)/Tz] × 100.
Experimental result: result is see table 4.
Table 4
Compound number is see test example 2.
Experimental result shows, the compounds of this invention is obvious to the effect of human breast carcinoma inhibiting tumour cells.
Test example 6 the compounds of this invention suppresses people's tumor cells of hepatocellular carcinoma propagation
Method: many people tumor cells of hepatocellular carcinoma Hep3B paves the end 96 orifice plate according to 2500 cells/well.In cultivating system, maximum drug concentration is 50 μMs, does drug level dilution according to 5 times of gradients.Drug effect adds 10 μ l CCk-8 after 48 hours, after hatching 6h, utilize microplate reader to measure 450nM wavelength absorption value.Drug on tumor inhibitory rate of cell growth computational methods are according to National Cancer Institute (National Cancer Institute, NCI) standard method is carried out: when Ti(medicine group cultivates 48h, CCK-8 colour developing absorbs OD value) >=Tz(CCK-8 colour developing absorption OD value when drug containing group is not cultivated initial), tumor cell survival=[(Ti-Tz)/(C-Tz)] × 100, wherein C be not drug containing group after 48 hours CCK-8 colour developing absorb OD value; As Ti<Tz, tumor cell survival=[(Ti-Tz)/Tz] × 100.
Experimental result: result is see table 5.
Table 5
Compound number is see test example 2.
Experimental result shows, the compounds of this invention is obvious to people's tumor cells of hepatocellular carcinoma inhibitory action.
Test example 7 the compounds of this invention suppresses the investigation of graft-rejection
Set up the skin transplantation model of individual mouse genotypes: select 8 ~ 12 weeks C57BL/6 and Bal b/c female mices to be experimental subject, raise in SPF level barrier environment, feedstuff irradiation sterilization, bedding and padding and the equal autoclaving of drinking water.With Balb/c mice for receptor, C57/BL/6 mice is donor.Prostrate fixing after donor anesthesia, about get the skin of back of 1cm × 1cm after sterilization, scrape off the tissues such as subcutaneous fat and blood vessel, be put in aseptic normal saline for subsequent use.Receptor anaesthetizes rear prostrate fixing, after the sterilization of back, takes off the skin more slightly bigger than donor dermal, as transplant bed.Donor dermal is laid on transplant bed, fixes with adhesive bandage after alignment.
Experiment is divided into 6 groups, often organizes 6 mices: model group (normal saline), positive controls (CyA, 5mg/kg/ days), the compounds of this invention 1,2,6,11 totally 4 groups (50mg/kg/ days) (compound number is see test example 2).Administration from transplanting the last week, successive administration one week again after transplanting, all adopts intraperitoneal injection.
Graft-rejection detection scheme: skin transplantation is dismantled after 7 days adhesive bandage, observes the color of skin grafts, hardness every day, comes off and Mus hair growing state, as hardening in skin graft or incrustation comes off, be then transplant rejection; Skin graft softness or the growth of Mus hair are then graft survival.Record skin graft, from being transplanted to the time occurring to repel, calculates skin graft mean survival time MST.Result is see Fig. 2.
Result shows, the compounds of this invention 1,2, and 6,11 all obviously can extend graft survival time, and action effect is suitable with positive control cyclosporin A, and the compounds of this invention effectively can suppress graft-rejection; Wherein, the drug action of compound 1 is better than other compounds.
Test example 8: invention compound suppresses delayed hypersensitivity (DTH).
Set up the mouse DTH model that dinitrofluorobenzene (DNFB) is induced: BALB/c mouse left and right sole even spread 20 μ l 0.5%(v/v) DNFB solution (being dissolved in 4:1 acetone-olive oil), with independent applied with acetone/olive oil solvent in contrast, every day 1 time, continuous 2 days.Within 9 days, provocative test is carried out: i.e. two sides even spread 10 μ l 0.5%(v/v inside and outside mouse right ear after 2nd sensitization) DNFB solution.Makes ear device with 8mm ear is swollen after 72h, auricle is got in ears punching, weighs and calculates the weight of auris dextra sheet increase.Animal divides into groups: 35 BALB/c mouse are divided into A, B, C, D, E, F group at random, often organizes 5 mices.A group is positive group of DNFB sensitization, and B group is positive control medicine CsA.C, D, E, F group is respectively the compounds of this invention 1, and 2,6,11 processed group (compound number is see test example 2).From the 1st DNFB sensitization a few days ago to getting auricle weighs, according to the dosage of 50mg/kg/ days, lumbar injection the compounds of this invention, positive control medicine CsA according to the dosage of 5mg/kg/ days, lumbar injection.Result is see Fig. 3.
Result shows, the compounds of this invention 1,2, and 6,11 all effectively can suppress delayed hypersensitivity.
Test example 9: the compounds of this invention suppresses rheumatoid arthritis
Select 6-10 week DBA/1J mice, by subcutaneous injection after 50ug cattle II Collagen Type VI and the complete emulsifying of equal-volume complete Freund's adjuvant (CFA).After 21 days with 50ug same antigen and incomplete Freund's adjuvant (IFA) fully emulsified after, booster immunization 1 time.Observed and recorded from the 45th day.Adopt 1-4 scoring method: 1 point, normally; 2 points, 1 arthroncus; 3 points, the arthroncus more than 1, but do not accumulate whole joint; 4 points, the serious swelling or tetanic of whole pawl.The scoring of every pawl is added the overall score namely obtaining mouse arthritis disease.The mice that joint overall score is greater than 1 is that model is successfully established.
Experiment grouping: normal male Balb/C mouse 6, successfully sets up the male DBA/1J mice 30 of mice rheumatoid arthritis model, often organizes 6, and only, grouping situation is as follows: A. normal mouse matched group for body weight 30 ± 4.7g/, B.CIA: saline therapy group (negative control group), CyA, 15mg/kg/day), the compounds of this invention 1:2-(1 hydrogen-benzothiazolyl-2 base)-4, 5, 6, 7-tetrahydrochysene-2 hydrogen-indazole-3-alcohol, the compounds of this invention 2:2-(1 hydrogen-benzimidazolyl-2 base)-4, 5, 6, 7-tetrahydrochysene-2 hydrogen-indazole-3-alcohol, the compounds of this invention 6:1-(2-[4-morpholinodithio base)-3-(trifluoromethyl)-1H-pyrazoles-5-alcohol, the compounds of this invention 11:2-(4-phenyl-2-thiazolyl)-4, 5, 6, 7-tetrahydrochysene-2H-indazole-3-alcohol administration group (50mg/kg/day).The 45th day first day for treatment, selects to carry out intramuscular injection inside mouse hind leg.Administration, after 2 weeks, is marked to each group of mouse arthritis disease.Result is see table 6.
Table 6
Grouping Scoring
Control 1
0.9%NaCl(feminine gender is according to group) 4
Cyclosporin A (positive control) 2
The compounds of this invention 1 administration group 2
The compounds of this invention 2 administration group 2
The compounds of this invention 6 administration group 3
The compounds of this invention 11 administration group 1
Compound number is see test example 2.
Experimental result shows, the compounds of this invention 1,2,6,11 effectively can treat rheumatoid arthritis, and its drug effect is suitable with positive control cyclosporin A; Wherein, the drug action of compound 11 is the strongest, is even better than positive drug.
In sum; the compounds of this invention can effective Profilin kinase activity; the T cell of abnormal activation can be effectively suppressed to be bred; can be used for disease such as treatment autoimmune disease, malignant tumor etc.; particularly lymphoma, multiple myeloma, breast carcinoma, hepatocarcinoma, graft-rejection, allergy and rheumatoid arthritis are had significant therapeutic effect, there is good prospects for commercial application.

Claims (3)

1. compound as follows purposes in the medicine preparing treatment malignant tumor or autoimmune disease,
Described compound is formula II compound, 2-(4-phenyl-2-thiazolyl)-4,5,6,7-tetrahydrochysene-2H-indazole-3-alcohol or 2-(6-methoxyl group-2-[4-morpholinodithio base)-4,5,6,7-tetrahydrochysene-2H-indazole-3-alcohol; Wherein, the structural formula of formula II compound is as follows
R 1for-OH ,-H, or-OR 7, described R 7for the alkyl of C1-C3;
R 2for alkyl or the-R of-H, C1-C5 8oH, described R 8for the alkyl of C1-C3;
R 3for the alkyl of the C1-C3 of the alkyl of-H, C1-C3, phenyl or halogen substiuted;
Or, R 2, R 3the cycloalkyl of C3-C8 is formed together with the carbon atom that they connect;
R 4for N, O or S;
Described malignant tumor is multiple myeloma, lymphocytic chronic myeloid leukemia, hepatocarcinoma or breast carcinoma;
Described autoimmune disease is rheumatoid type arthritis, graft-rejection or allergy.
2. purposes according to claim 1, is characterized in that:
R 1for-OH, or-OR 7, described R 7for methyl;
R 2for-H or-R 8oH, described R 8for ethyl;
R 3for the methyl that methyl, phenyl or trifluoro replace;
Or, R 2, R 3the cycloalkyl of C5-C6 is formed together with the carbon atom that they connect;
R 4for N, O or S.
3. purposes according to claim 1, is characterized in that: described compound is:
2-(1 hydrogen-benzothiazolyl-2 base)-4,5,6,7-tetrahydrochysene-2 hydrogen-indazole-3-alcohol,
2-(1 hydrogen-benzimidazolyl-2 base)-4,5,6,7-tetrahydrochysene-2 hydrogen-indazole-3-alcohol,
1-(1,3-benzothiazolyl-2 base)-1,5,6,7-tetrahydrochysene-4H-indazole-4-ketone,
1-(1,3-benzothiazolyl-2 base)-3,4-dimethyl-1 hydrogen-pyrazoles-5-alcohol,
2-(2-[4-morpholinodithio base)-2,4,5,6-tetrahydro cyclopentyl pyrazoles-3-alcohol,
1-(2-[4-morpholinodithio base)-3-(trifluoromethyl)-1H-pyrazoles-5-alcohol,
1-(2-[4-morpholinodithio base)-4-(2-ethoxy)-3-methyl isophthalic acid H-pyrazoles-5-alcohol,
2-(2-[4-morpholinodithio base)-4,5,6,7-tetrahydrochysene-2H-indazole-3-acetate,
2-(2-benzoxazolyl)-4,5,6,7-tetrahydrochysene-2H-indazole-3-alcohol or
1-(2-[4-morpholinodithio base)-3-phenyl-1H-pyrazoles-5-alcohol.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1030757A (en) * 1987-06-17 1989-02-01 伟材株式会社 Benzothiazole derivant
CN101686964A (en) * 2007-05-22 2010-03-31 威斯康星旧生研究基金会 The anti-bacterial drug targeting of genome maintenance interfaces

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